This project will test the hypothesis that defects in DNA repair are acquired during stages of melanoma development and produce a chromosomal-mutator phenotype in UV-irradiated cells. Focus will be on nucleotide excision repair (NER), post-replication repair (PRR), DNA double-strand break (dsb) repair and intra-S checkpoint responses to the component of sunlight (UV radiation) that is most efficient in inducing DNA damage. In the three specific aims, DNA repair will be quantified in strains of normal human melanocytes, in melanoma cell lines, and in melanocytes expressing mutated genes commonly found in melanoma. Specific Aim 1 will determine whether NER capacity is attenuated or lost during stages of melanomagenesis. PRR capacity (translesions synthesis and gap repair) and the intra-S checkpoint response of inhibition of replicon initiation will be measured in Specific Aim 2. Proposed studies will also investigate the hypothesis that UV-induced DNA damage triggers a signaling pathway that results in trans-inhibition of DNA chain elongation;this novel element of the intra-S checkpoint is postulated to reduce the rate of fork displacement in replicons that have not yet encountered a template lesion. A DNA fiber-spreading and immuno-staining assay will enable visualization of replication dynamics and measurement of individual replication tracks. Knockdown of the Timeless-interacting protein (Tipin), together with ectopic expression of RNAi-resistant Tipin, will test whether this protein regulates the rate of displacement of DNA replication forks through its binding to RPA. Induction of chromosomal aberrations and INK4a allelic deletions in UV-treated cells will be examined in Specific Aim 3. Aberrations and deletions are thought to be associated with DNA dsb generated at collapsed replication forks and other single-strand DNA regions formed during replication of the UV-damaged DNA. Phospho-histone H2AX/phospho-ATM/Mre11-positive nuclear foci will be quantified to monitor the formation and repair of UV-induced DNA dsb. Studies will determine whether genetic alterations associated with melanoma development enhance UV-clastogenesis in cultured melanocytes. The proposed studies will shed light on how an environmental carcinogen, sunlight, induces skin cancer by expanding our knowledge of DNA damage responses in normal human melanocytes and providing insight into the mechanisms of genetic instability in melanoma.